P849: DEVELOPMENT OF A QUANTITATIVE ASSAY FOR ASSESSING BCL2’S PPI COMPLEXES AND ITS APPLICATION AS A TARGET ENGAGEMENT ASSAY IN CLINICAL SPECIMENS WITH MULTIPLE MYELOMA
Authors: Hongwon Lee, Byungsan Choi, Saem Hong, Yunseo Lee, Hayoung Park, Ja Min Byun, Youngil Koh, Tae-Young Yoon
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: The protein-protein interactions (PPIs) between members of the BCL2 family is the underlying mechanism for the regulation of the apoptosis pathway. In various types of cancers, cancer cells evade apoptosis by upregulating anti-apoptotic proteins like BCL2. The development of PPI inhibitors for BCL2 family proteins has enabled selective disruption of the target anti-apoptotic proteins’ PPIs, leading to the initiation of apoptosis signaling in cancers. As a result, an assessment of changes in BCL2 family PPI complexes following the administration of selective PPI inhibitor facilitates the evaluation of the potency and efficacy of BCL2-targeted anti-cancer inhibitors. Aims: The quantification of BCL2 PPI complexes, particularly in clinical specimens, has been limited due to a lack of suitable sensitivity for measuring PPI complexes from small amounts of clinical samples. Therefore, the development of a sensitive method for quantifying BCL2 PPI complexes in clinical specimens would be of great benefit in evaluating the effectiveness of BCL2-targeted anti-cancer agents. Methods: We describe a single-molecule co-immunoprecipitation (co-IP)-based PPI complex profiling method that quantifies the level of BCL2 PPI complexes (Fig. 1A). From BMMCs of 4 patients with multiple myeloma, we assessed BCL2 PPI complexes with various doses of ABT-199 treatment. We applied 5 assays for measuring PPI complexes and expression levels related to BCL2 protein. Concurrently, in vitro ABT-199 sensitivity of these samples was determined by staining with Annexin V serving as a marker for early apoptosis. Results: Our developed method was capable of measuring 5 different metrics related to BCL2 protein from 100,000 BMMCs. The levels of BCL2 PPI complexes prior to treatment with ABT-199 varied greatly among patients (Fig. 1B and C, dose at DMSO). We also found that the amounts of BCL2 PPI complexes were not simple function of the level of BCL2 protein (Fig. 1B and C, BCL2-BIM versus BCL2-BAX). ABT-199 treatment disrupted the BCL2 PPI complexes in a dose dependent manner, supporting the molecular function of ABT-199 in interfering with the PPI of BCL2. Interestingly, the BMMC that showed the highest level of BCL2-BAX PPI complex responded to in vitro ABT-199 treatment more than other BMMCs, whereas the level of BCL2-BIM PPI complex was largely similar among them. Collectively, these results indicated that the level of BCL2-BAX PPI complex prior to drug treatment could be a potential biomarker for predicting ABT-199 response in multiple myeloma.Summary/Conclusion: We developed a platform that determines the amounts of BCL2’s PPI complexes in a quantitative manner. Our platform requires 100,000 cells for 5 assays related to BCL2, making it applicable to the analysis of clinical samples that are typically limited in volume and cell count. Using our platform, we revealed that patient samples can carry widely varying amounts of BCL2 proteins and PPI complexes, which are often closely related to their sensitivity to a BCL2 inhibitor. The project OPTIMUS launched by the US FDA encourages pharmaceutical companies to optimize the dose of targeted drug compounds for efficacy, rather than using the traditional maximum tolerated dose (MTD). Thus, determination of the most effective dose for the targeted drug compound is critical during clinical trials. When applied to clinical cohorts in dose escalation studies, our platform could help determine the optimal dose of the BCL2 inhibitor, preventing patients from being exposed to toxicities from unnecessarily high drug doses. Keywords: BCL-XL, Apoptosis, BCL2